This invention relates to the field of diagnostics based on DNA sequence information.
Antimicrobial resistance to multiple antibiotics is a significant and well described clinical problem; however, a less well-characterized phenomenon, antimicrobial tolerance, has emerged in pathogenic isolates of Streptococcus pneumoniae with potentially serious effects on patient outcome. Tolerance describes the ability of bacteria to stop growing in the presence of an antibiotic, while surviving to resume growth once the antibiotic is remove; Incidence of tolerance to vancomycin, the antibiotic of last resort for Gram-positive infections, has increased to 8% in the past few years. Tolerance has also been implicated in-poor patient outcome with pneumococcal meningitis, mortality 30% versus non-tolerant 5% (unpublished data).
In 1997, a locus was identified that is believed to control the activation of the major pneumococcal autolytic enzyme LytA, which is the enzyme whose loss of function is associated with tolerance. Novak R. B. et al, xe2x80x9cEmergence of vancomycin tolerance in Streptococcus pneumoniaexe2x80x99, Nature 399:590-593 (1999). The operon, vex/pep27/vncr/s, encodes for a signal peptide, Pep27, that is transported out of the cell via the Vex dedicated transporter. Novak, R. et al., xe2x80x9cSignal transduction by a death signal peptide: uncovering the mechanism of bacterial killing by penicillinxe2x80x9d, Molec Cell. 5:49-57 (2000). Pep27 is believed to be a quorum sensing peptide. Novak et al., id. (2000). Once it reaches a critical density in the supernatant, it signals through the two-component regulatory system, VncS and VncR, which subsequently induces activation of LytA. Novak et al., id. (2000).
It has been demonstrated that mutating any one of the genes of the vex/pep27/vncr/s operon prevents proper signaling, resulting in lack of LytA activation and tolerance to penicillin and vancomycin. Novak et al., id. (2000). However, the genetic basis for naturally occurring vancomycin tolerance in the community has not been determined.
The present invention provides a rapid diagnostic assay to identify strains of bacteria, particularly strains of Streptococcus pneumoniae, which are likely to have acquired tolerance to antibiotics. The assay is based on the identification of allelic variations with the vncS, vex2 and pep27 genes that are closely associated with tolerance to penicillin and vancomycin when present.
In one aspect, combinations of vex2 and pep27 alleles which are associated with antibiotic tolerance are taught.
In another aspect, an allele of the vncS gene which is associated with antibiotic tolerance is taught.
Single nucleotide polymorphisms (SNPs) which identify the various vncS, vex2 and pep27 alleles which are relevant to the present invention are taught. According to the present invention, the likely presence of antibiotic tolerant bacterial strains can be identified by determining the presence or absence of these distinctive SNPs in the vncS, vex2 and pep27 genes that are associated with antibiotic tolerance.
Various methods for identifying the SNPs taught herein or and kits providing the components needed to perform such methods are included herein as part of the invention.
SEQ ID No. 1 is a portion of the Type 4 allele of the vex2 gene. The presence of a xe2x80x9cGxe2x80x9d nucleotide at position 41 and an xe2x80x9cAxe2x80x9d nucleotide at position 67 identify this as a Type 4 allele.
SEQ ID No. 2 is a portion of the R6 allele of the vex2 gene. The presence of an xe2x80x9cAxe2x80x9dnucleotide at position 41 and a xe2x80x9cGxe2x80x9d nucleotide at position 67 identify this as an R6 allele.
SEQ ID No. 3 is a portion of the Type 4 allele of the pep27 gene. The presence of a xe2x80x9cGxe2x80x9d nucleotide at position 35 and a xe2x80x9cGxe2x80x9d nucleotide at position 46 identify this as a Type 4 allele.
SEQ ID No. 4 is a portion of the R6 allele of the pep27 gene. The presence of an xe2x80x9cAxe2x80x9d nucleotide at position 35 and an xe2x80x9cAxe2x80x9d nucleotide at position 46 identify this as an R6 allele.
SEQ ID No. 5 is a portion of the wildtype allele for the vncS gene. The presence of a xe2x80x9cTxe2x80x9d nucleotide at position 79 identifies this as a wildtype allele.
SEQ ID No. 6 is a portion of the vancomycin tolerant allele for the vncS gene. The presence of a xe2x80x9cCxe2x80x9d nucleotide at position 79 identifies this as a vancomycin tolerant allele.
SEQ ID No. 7 is a forward primer for the vex2 gene which hybridizes to a region approx. 255 nucleotides upstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No. 8 is a reverse primer for the vex2 gene which hybridizes to a region approx. 160 nucleotides downstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No.9 is a forward primer for the pep27 gene which hybridizes to a region about 90 nucleotides upstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No. 10 is a reverse primer for the pep27 gene which hybridizes to a region about 90 nucleotides downstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No. 11 is a forward primer for the vncS gene which hybridizes to a region about 380 nucleotides upstream of the SNP which distinguishes the wildtype allele from the antibiotic tolerant allele.
SEQ ID No. 12 is a reverse primer for the vncS gene which hybridizes to a region about 30 nucleotides downstream of the SNP which distinguishes the wildtype allele from the antibiotic tolerant allele.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, xe2x80x9cMolecular Cloning: A Laboratory Manualxe2x80x9d (1989); xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d Volumes I-III [Ausubel, R. M., ed. (1994)]; xe2x80x9cCell Biology: A Laboratory Handbookxe2x80x9d Volumes I-III [J. E. Celis, ed. (1994))]; xe2x80x9cCurrent Protocols in Immunologyxe2x80x9d Volumes I-III [Coligan, J. E., ed. (1994)]; xe2x80x9cOligonucleotide Synthesisxe2x80x9d (M. J. Gait ed. 1984); xe2x80x9cNucleic Acid Hybridizationxe2x80x9d [B. D. Hames and S. J. Higgins eds. (1985)]; xe2x80x9cTranscription And Translationxe2x80x9d [B. D. Hames and S. J. Higgins, eds. (1984)]; xe2x80x9cAnimal Cell Culturexe2x80x9d [R. I. Freshney, ed. (1986)]; xe2x80x9cImmobilized Cells And Enzymesxe2x80x9d [IRL Press, (1986)]; B. Perbal, xe2x80x9cA Practical Guide To Molecular Cloningxe2x80x9d (1984).
Definitions: The terms and phrases used herein to describe and claim the present invention shall have the meanings set forth below.
By xe2x80x9cantibiotic tolerancexe2x80x9d or xe2x80x9cantibiotic tolerantxe2x80x9d is meant the ability of bacteria to stop growing in the presence of an antibiotic, while surviving to resume growth once the antibiotic is removed. In contrast to antibiotic resistance, bacterial strains which are antibiotic tolerant cannot be killed by increasing the amount of antibiotic used.
By xe2x80x9coligonucleotide,xe2x80x9d is meant a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The oligonucleotides of the invention are preferably from 10 to 50 nucleotides in length, even more preferably from 20-30 nucleotides in length or from 15-25 nucleotides in length, and may be DNA, RNA or synthetic nucleic acid, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be appreciated by those skilled in the art. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence to form a stable hybrid. Such molecules are known in the art and include, for example, peptide nucleic acids (PNAs) in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
By xe2x80x9cprimerxe2x80x9d is meant an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 10 or more nucleotides, preferably 15-25 nucleotides, although it may contain fewer nucleotides or more nucleotides.
The primers herein are selected to be xe2x80x9csubstantiallyxe2x80x9d complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5xe2x80x2 end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
A labeled oligonucleotide or primer may be utilized in the methods, assays and kits of the present invention. The labeled oligonucleotide may be utilized as a primer in PCR or other method of amplification and may be utilized in analysis, as a reactor or binding partner of the resulting amplified product. In certain methods, where sufficient concentration or sequestration of the subject nucleic acid has occurred, and wherein the oligonucleotide label and methods utilized are appropriately and sufficiently sensitive, the nucleic acid may be directly analyzed, with the presence of, or presence of a particular label indicative of the result and diagnostic of the presence or absence of a particular vex2, pep27 or vncS allele. After the labeled oligonucleotide or primer has had an opportunity to react with sites within the sample, the resulting product may be examined by known techniques, which may vary with the nature of the label attached. The label utilized may be radioactive or non-radioactive, including fluorescent, colorimetric or enzymatic. In addition, the label may be, for instance, a physical or antigenic tag which is characterized by its activity or binding.
In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re are used, known currently counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
By xe2x80x9csubjectxe2x80x9d is meant a human or animal which is susceptible to infection by a bacteria which is normally treatable with antibiotics such as penicillin or vancomycin, but which can be antibiotic tolerant if it contains a tolerant form of the vncS allele or a combination of vex2 and pep27 alleles that confers antibiotic tolerance as taught herein.
By xe2x80x9crestriction enzymexe2x80x9d is meant a bacterial enzyme which cleaves double-stranded DNA at or near a specific nucleotide sequence referred to as a restriction enzyme recognition site.
Bacterial infections may be treated with a variety of antibiotics. When considering whether to treat an infection with an antibiotic and which antibiotic to use, it is important to know as much as possible about the bacteria causing the infection. In particular, it is very helpful to know in advance if the infection includes a population of bacteria which are tolerant to a particular antibiotic. This knowledge can avoid treatment of infections with antibiotics which will not be effective or treatments that are too short in duration to effectively kill tolerant strains.
The present invention provides methods for rapidly identifying bacteria which are likely to be tolerant to certain antibiotics and which therefore cannot be effectively treated using such antibiotics. These methods are based on the identification of the genetic basis of antibiotic tolerance that has been found in naturally occurring bacterial strains.
The present invention teaches specific allelic variations within the vncS, vex2 and pep27 genes that are closely associated with antibiotic tolerance when present. The variation within the vncS gene that confers tolerance occurs in the region of the vncS gene shown in SEQ ID No. 5. Within this region, the tolerant vncS allele is identified by a change from a thymidine to a cytosine at position 79 of SEQ ID Nos. 5 and 6.
According to the present invention, antibiotic tolerance is also associated with a combination of vex2 and pep27 alleles. Both the vex2 and pep27 genes have alleles designated as xe2x80x9cType 4xe2x80x9d and xe2x80x9cR6xe2x80x9d. As taught herein, a specific mismatch between vex2 and pep27 alleles is associated with antibiotic tolerance. Specifically, a bacteria having a combination of a Type 4 vex 2 allele and an R6 pep27 allele is classified as highly likely to be antibiotic tolerant according to the present invention.
The variation that identifies a specific vex2 gene as either a Type 4 or R6 allele is shown in SEQ ID Nos 1 and 2. The Type 4 allele has a guanosine at position 41 and an adenosine at position 67 of SEQ ID No. 1 while the R6 allele has an adenosine and a guanosine at these respective positions (see SEQ ID No. 2).
The variation that identifies a specific pep27 gene as either a Type 4 or R6 allele is shown in SEQ ID Nos. 3 and 4. The Type 4 allele has a guanosine at positions 35 and 46 of SEQ ID No. 3 while the R6 allele has an adenosine at both of these positions (see SEQ ID No. 4).
A bacterial strain which has either (a) the tolerant allele of the vncS gene (SEQ ID No. 6), (b) the specific allelic mismatch between the vex2 and pep27 genes, or (c) both (a) and (b) is identified as likely to be an antibiotic tolerant strain according to the invention.
The allelic variations taught herein as associated with antibiotic tolerance may be identified in the DNA of a selected bacteria using any desired means. Such means includes various conventional techniques for sequencing the critical regions of vex2, pep27 and vncS genes which identify tolerant alleles/allele combinations including pyrosequencing, various conventional polymerase chain reaction (PCR) techniques that can be adapted to detect SNPs such as real time PCR, Invader(trademark) Technology (Third Wave Technologies;Lyamichev et al., Nature Biotech. 17: 292-296 (March 1999), Luminex 11(trademark) technology (Iannone et al., Cytometry 39: 131-140 (2000), NanoChip(trademark) Technology (Gilles et al., Nature Biotech. 17: 365-370 (April 1999).
As indicated, the polymorphisms which distinguish the alleles in these regions may be identified using polymerase chain reaction (PCR) technology and appropriate primers. Appropriate primers will be designed to amplify regions encompassing the polymorphisms identified herein. Primers may be designed such that, depending on the nucleotide present at the site of the polymorphism, a restriction enzyme recognition site may be created in the amplified fragment. In this case the identity of the nucleotide present at the site of the polymorphism can be determined by testing the amplified fragment for cleavage with the appropriate restriction enzyme.
In a further embodiment of this invention, test kits may be prepared to determine the genotype of a selected bacterial strain with respect to the vex2, pep27 and vncS genes, and thereby determine whether the strain is likely to be antibiotic tolerant.
Accordingly, a test kit may be prepared for determining whether a selected bacteria has a combination of vex2 and pep27 alleles associated with antibiotic tolerance. Further, a test kit may be prepared for determining whether a selected bacteria has an allele of the vncS gene associated with antibiotic tolerance. The test kit will include components for the PCR amplification and analysis of critical regions of the vex2, pep27 and/or vncS genes. In one embodiment, the critical region is amplified and then directly sequenced. In an additional embodiment, the critical region is amplified and its sequence at the polymorphic locations identified herein is determined by assessing susceptibility of the PCR product to cleavage with a particular restriction enzyme or a set of restriction enzymes. In a further embodiment, specific primer sets are utilized in amplification of the critical region and the presence or absence of PCR product with the specific primer sets is evaluated to determine the sequence at the polymorphic locations identified herein. Additional reagents for PCR amplification and evaluation may be included in the kit along with directions for use.
In a further aspect, the present invention provides oligonucleotide primers or probes suitable for use in the determination of the vex2, pep27 or vncS genotypes of a selected bacterial strain. Oligonucleotide primers preferably hybridize to a region within 500 nucleotides of the SNP of interest, more preferably within 100 nucleotides of the SNP.
In a particular embodiment, the present invention relates to an isolated oligonucleotide primer having a sequence selected from the group consisting of
The diagnostic and therapeutic utility of the present invention extends to the use of the assays, methods and kits of the present invention in determining whether a subject suffering from a bacterial infection can be effectively treated with antibiotics, particularly penicillin and vancomycin. Samples of the infecting bacteria can be rapidly assayed using the methods of the invention. The information thus obtained from the methods of the present invention can then be used to help predict whether the subject can be successfully treated with a particular antibiotic. For example, if it is determined using the methods of the present invention that a subject is infected with bacteria having a tolerant allele of the vncS gene and/or a mismatch of vex2 and pep27 alleles associated with tolerance as taught herein, it would be predicted that this subject could not be successfully treated with antibiotics such as penicillin or vancomycin. In this case unsuccessful treatment of the subject with these conventional antibiotics could be avoided and alternative therapies could be administered without delay.
The present invention may be better understood by reference to the following non-limiting examples. These examples are presented in order to more fully illustrate the invention through the description of particular embodiments. These examples should in no way be construed as limiting the scope of the invention.